MicroRNAs are key regulators of many biological processes, including cell differentiation. Here we show that during human monocyte-macrophage differentiation, expression of the microRNAs miR-223, miR-15a and miR-16 decreased considerably, which led to higher expression of the serine-threonine kinase IKKalpha in macrophages. In macrophages, higher IKKalpha expression in conjunction with stabilization of the kinase NIK induced larger amounts of p52. Because of low expression of the transcription factor RelB in untreated macrophages, high p52 expression repressed basal transcription of both canonical and noncanonical NF-kappaB target genes. However, proinflammatory stimuli in macrophages resulted in greater induction of noncanonical NF-kappaB target genes. Thus, a decrease in certain microRNAs probably prevents macrophage hyperactivation yet primes the macrophage for certain responses to proinflammatory stimuli.

Chronic obstructive pulmonary disease is characterized by emphysema, chronic bronchitis, and small airway remodeling. The alveolar destruction associated with emphysema cannot be repaired by current clinical practices. Stem cell therapy has been successfully used in animal models of cigarette smoke- and elastase-induced emphysema. However, the optimal dose of mesenchymal stem cells (MSCs) for the most effective therapy has not yet been determined. It is vital to determine the optimal dose of MSCs for clinical application in emphysema cases.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. High mortality from HCC is mainly due to widespread prevalence and the lack of effective treatment, since systemic chemotherapy is ineffective, while the targeted agent Sorafenib extends median survival only briefly. The steroidal saponin 20(S)-ginsenoside Rg3 from Panax ginseng C.A. Meyer is proposed to chemosensitize to various therapeutic drugs through an unknown mechanism. Since autophagy often serves as cell survival mechanism in cancer cells exposed to chemotherapeutic agents, we examined the ability of Rg3 to inhibit autophagy and chemosensitize HCC cell lines to doxorubicin in vitro. We show that Rg3 inhibits late stage autophagy, possibly through changes in gene expression. Doxorubicin-induced autophagy plays a protective role in HCC cells, and therefore Rg3 treatment synergizes with doxorubicin to kill HCC cell lines, but the combination is relatively nontoxic in normal liver cells. In addition, Rg3 was well-tolerated in mice and synergized with doxorubicin to inhibit tumor growth in HCC xenografts in vivo. Since novel in vivo inhibitors of autophagy are desirable for clinical use, we propose that Rg3 is such a compound, and that combination therapy with classical chemotherapeutic drugs may represent an effective therapeutic strategy for HCC treatment.

Mesenchymal stem cells (MSCs) effectively reduce airway inflammation and regenerate the alveolus in cigarette- and elastase-induced chronic obstructive pulmonary disease (COPD) animal models. The effects of stem cells are thought to be paracrine and immune-modulatory because very few stem cells remain in the lung one day after their systemic injection, which has been demonstrated previously. In this report, we analyzed the gene expression profiles to compare mouse lungs with chronic exposure to cigarette smoke with non-exposed lungs. Gene expression profiling was also conducted in a mouse lung tissue with chronic exposure to cigarette smoke following the systemic injection of human cord blood-derived mesenchymal stem cells (hCB-MSCs). Globally, 834 genes were differentially expressed after systemic injection of hCB-MSCs. Seven and 21 genes, respectively, were up-and downregulated on days 1, 4, and 14 after HCB-MSC injection. The Hbb and Hba, genes with oxygen transport and antioxidant functions, were increased on days 1 and 14. A serine protease inhibitor was also increased at a similar time point after injection of hCB-MSCs. Gene Ontology analysis indicated that the levels of genes related to immune responses, metabolic processes, and blood vessel development were altered, indicating host responses after hCB-MSC injection. These gene expression changes suggest that MSCs induce a regeneration mechanism against COPD induced by cigarette smoke. These analyses provide basic data for understanding the regeneration mechanisms promoted by hCB-MSCs in cigarette smoke-induced COPD.

Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a central protein in necroptosis, but posttranslational processes that regulate RIP3 activity and stability remain poorly understood. Here, we identify pellino E3 ubiquitin protein ligase 1 (PELI1) as an E3 ligase that targets RIP3 for proteasome-dependent degradation. Phosphorylation of RIP3 on T182 leads to interaction with the forkhead-associated (FHA) domain of PELI1 and PELI1-mediated K48-linked polyubiquitylation of RIP3 on K363. This same phosphorylation event is also important for RIP3 kinase activity; thus, PELI1 preferentially targets kinase-active RIP3 for degradation. PELI1-mediated RIP3 degradation effectively prevents cell death triggered by RIP3 hyperactivation. Importantly, upregulated RIP3 expression in keratinocytes from toxic epidermal necrolysis (TEN) patients is correlated with low expression of PELI1, suggesting that loss of PELI1 may play a role in the pathogenesis of TEN. We propose that PELI1 may function to control inadvertent activation of RIP3, thus preventing aberrant cell death and maintaining cellular homeostasis.

The At-rich interactive domain 1A (ARID1A) is frequently mutated in gastric cancers (GCs) with a poor prognosis. Growing evidence indicates that loss of ARID1A expression leads to activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway by AKT phosphorylation. We aim to investigate the different sensitivity for the AKT inhibitor in ARID1A-deficient GC cells.

Mixed lineage kinase domain-like (MLKL) is an essential molecule of necroptosis, a cell death process that is initiated by direct disruption of the plasma membrane. During necroptosis, MLKL is phosphorylated by receptor interacting protein kinase-3 (RIPK3 or RIP3), and then translocates to the plasma membrane to disrupt membrane integrity. Recent data suggest that MLKL also has a RIP3-indendent function in the generation of intraluminal and extracellular vesicles (EVs), as well as in myelin sheath breakdown when promoting sciatic nerve regeneration. Here we show that depletion of MLKL enhances TRAIL-induced cell death in a RIP3-independent manner. Depletion of MLKL leads to prolonged cytotoxic signals that increase TRAIL-induced cell death. Initially, TRAIL binds to DR5 at the cell surface and is endocytosed at similar rates in MLKL-expressing and MLKL-depleted cells, eventual degradation of intracellular TRAIL by the lysosome is delayed in MLKL-depleted cells, corresponding with prolonged/enhanced intracellular signals such as p-ERK and p-p38 in these cells. Colocalization of TRAIL with the marker of early endosomes, EEA1 suggests that TRAIL is accumulated in early endosomes in MLKL-depleted cells compared to MLKL-expressing cells. This indicates that depletion of MLKL reduces receptor-ligand endosomal trafficking leading to increased TRAIL-cytotoxicity. An MLKL mutant that compromises its necroptotic function and its function in the generation of EVs was sufficient to rescue MLKL deficiency, suggesting that the N-terminal structural elements necessary for these functions are not required for the function of MLKL in the intracellular trafficking associated with regulating death receptor cytotoxicity. A reduction in MLKL expression in cancer cells would therefore be expected to result in enhanced TRAIL-induced therapeutic efficacy.

miRNA (miR)-4742-5p is a recently identified microRNA regarding progression and metastasis in gastric cancer (GC). However, the biological function of this novel miRNA is largely unknown. We identified that the miR-4742-5p expression level was variably increased in GC cell lines. Suppression of miR-4742-5p using miR-inhibitor reduced the proliferation, migration, and invasion of GC cells with high miR-4742-5p expression, whereas overexpression of miR-4742-5p-mimic enhanced the aforementioned properties in GC cells with low miR-4742-5p expression. miR-4742-5p expression induced the decreases of Zo-1 and E-cadherin expression as well as the increases of vimentin and N-cadherin expression, leading to epithelial-mesenchymal transition (EMT) of cancer cells. RNA sequencing results indicated Ras-related GTP-binding protein 43 (Rab43) as a potential target gene. We identified that the expression of Rab43 is associated with activation of AKT and nuclear factor-kappa B (NF-κB) which are key oncogenic pathways in cancer cells. Our results demonstrate a new component in GC progression, promising a potential therapeutic strategy.

Activating transcription factor 3 (ATF3) is a negative regulator of proinflammatory cytokine expression in macrophages, and ATF3 deficient mice are more susceptible to endotoxic shock. This study addresses the role of ATF3 in the Kdo(2)-Lipid A-induced Toll-like receptor 4 (TLR4) signaling pathway in mouse embryonic fibroblasts (MEF). Kdo(2)-Lipid A upregulates ATF3 expression in wild type MEF cells and induces both nuclear factor kappa B (NF-κB) and c-Jun N-terminal kinase (JNK) activation via the TLR4 signaling pathway, while neither of these pathways is activated in ATF3-/- MEF cells. Interestingly, in contrast to Kdo(2)-Lipid A, the activation of both NF-κB and JNK by TNF-α was normal in ATF3-/- MEF cells.

Generally, cancer stem cells have epithelial-to-mesenchymal-transition characteristics and other aggressive properties that cause metastasis. However, there have been no confident markers for the identification of cancer stem cells and comparative methods examining adherent and sphere cells are widely used to investigate mechanism underlying cancer stem cells, because sphere cells have been known to maintain cancer stem cell characteristics. In this study, we conducted a meta-analysis that combined gene expression profiles from several studies that utilized tumorsphere technology to investigate tumor stem-like breast cancer cells. We used our own gene expression profiles along with the three different gene expression profiles from the Gene Expression Omnibus, which we combined using the ComBat method, and obtained significant gene sets using the gene set analysis of our datasets and the combined dataset. This experiment focused on four gene sets such as cytokine-cytokine receptor interaction that demonstrated significance in both datasets. Our observations demonstrated that among the genes of four significant gene sets, six genes were consistently up-regulated and satisfied the p-value of < 0.05, and our network analysis showed high connectivity in five genes. From these results, we established CXCR4, CXCL1 and HMGCS1, the intersecting genes of the datasets with high connectivity and p-value of < 0.05, as significant genes in the identification of cancer stem cells. Additional experiment using quantitative reverse transcription-polymerase chain reaction showed significant up-regulation in MCF-7 derived sphere cells and confirmed the importance of these three genes. Taken together, using meta-analysis that combines gene set and network analysis, we suggested CXCR4, CXCL1 and HMGCS1 as candidates involved in tumor stem-like breast cancer cells. Distinct from other meta-analysis, by using gene set analysis, we selected possible markers which can explain the biological mechanisms and suggested network analysis as an additional criterion for selecting candidates.

Mesenchymal stem cells (MSCs) obtained from bone marrow or adipose tissue can successfully repair emphysematous animal lungs, which is a characteristic of chronic obstructive pulmonary disease. Here, we describe the cellular distribution of MSCs that were intravenously injected into mice with elastase-induced emphysema. The distributions were also compared to the distributions in control mice without emphysema.

Chromatin remodelers regulate the nucleosome barrier during transcription, DNA replication, and DNA repair. The chromatin remodeler RSF1 is enriched at mitotic centromeres, but the functional consequences of this enrichment are not completely understood. Shugoshin (Sgo1) protects centromeric cohesion during mitosis and requires BuB1-dependent histone H2A phosphorylation (H2A-pT120) for localization. Loss of Sgo1 at centromeres causes chromosome missegregation. Here, we show that RSF1 regulates Sgo1 localization to centromeres through coordinating a crosstalk between histone acetylation and phosphorylation. RSF1 interacts with and recruits HDAC1 to centromeres, where it counteracts TIP60-mediated acetylation of H2A at K118. This deacetylation is required for the accumulation of H2A-pT120 and Sgo1 deposition, as H2A-K118 acetylation suppresses H2A-T120 phosphorylation by Bub1. Centromeric tethering of HDAC1 prevents premature chromatid separation in RSF1 knockout cells. Our results indicate that RSF1 regulates the dynamics of H2A histone modifications at mitotic centromeres and contributes to the maintenance of chromosome stability.

Cell therapy using stem cells has produced therapeutic benefits in animal models of COPD. Secretory mediators are proposed as one mechanism for stem cell effects because very few stem cells engraft after injection into recipient animals. Recently, nanovesicles that overcome the disadvantages of natural exosomes have been generated artificially from cells. We generated artificial nanovesicles from adipose-derived stem cells (ASCs) using sequential penetration through polycarbonate membranes. ASC-derived artificial nanovesicles displayed a 100 nm-sized spherical shape similar to ASC-derived natural exosomes and expressed both exosomal and stem cell markers. The proliferation rate of lung epithelial cells was increased in cells treated with ASC-derived artificial nanovesicles compared with cells treated with ASC-derived natural exosomes. The lower dose of ASC-derived artificial nanovesicles had similar regenerative capacity compared with a higher dose of ASCs and ASC-derived natural exosomes. In addition, FGF2 levels in the lungs of mice treated with ASC-derived artificial nanovesicles were increased. The uptake of ASC-derived artificial nanovesicles was inhibited by heparin, which is a competitive inhibitor of heparan sulfate proteoglycan that is associated with FGF2 signaling. Taken together, the data indicate that lower doses of ASC-derived artificial nanovesicles may have beneficial effects similar to higher doses of ASCs or ASC-derived natural exosomes in an animal model with emphysema, suggesting that artificial nanovesicles may have economic advantages that warrant future clinical studies.

S100A8/A9 has been suggested as a marker of disease activity in patients with adult-onset Still's disease (AOSD). We evaluated the clinical significance of S100A8/A9 as a biomarker and its pathogenic role in AOSD. Blood samples were collected prospectively from 20 AOSD patients and 20 healthy controls (HCs). Furthermore, skin and lymph node biopsy specimens of AOSD patients were investigated for S100A8/A9 expression levels via immunohistochemistry. Peripheral blood mononuclear cells (PBMCs) of active AOSD patients and HCs were investigated for S100A8/A9 cell signals. S100A8/A9, interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) levels in active AOSD patients were higher than those of HCs. S100A8/A9 levels correlated positively with IL-1β, TNF-α and C-reactive protein. The inflammatory cells expressing S100A8/A9 were graded from one to three in skin and lymph node biopsies of AOSD patients. The grading for S100A8/A9 was more intense in the skin lesions with karyorrhexis, mucin deposition, and neutrophil infiltration. Like lipopolysaccharide (LPS), S100A8/A9 induced phosphorylation of p38 and c-Jun amino-terminal kinase (JNK) in PBMCs, suggesting that S100A8/A9 activates Toll-like receptor 4 signaling pathways. These findings suggest that S100A8/A9 may be involved in the inflammatory response with induction of proinflammatory cytokines and may serve as a clinicopathological marker for disease activity in AOSD.

There is no therapy currently available that influences the natural history of disease progression in patients with chronic obstructive pulmonary disease (COPD). Although stem cell therapy is considered a potential therapeutic option in COPD, there are no clinical trials proving definitive therapeutic effects in patients with COPD. Recently, it was reported that pioglitazone might potentiate the therapeutic effects of stem cells in patients with heart or liver disease. To test the capacity of pioglitazone pretreatment of stem cells for emphysema repair, we evaluated the therapeutic effects of pioglitazone-pretreated human adipose-derived mesenchymal stem cells (ASCs) on elastase-induced or cigarette smoke-induced emphysema in mice. We also investigated the mechanisms of action of pioglitazone-pretreated ASCs. Pioglitazone-pretreated ASCs had a more potent therapeutic effect than non-pretreated ASCs in the repair of both elastase-induced and smoke-induced emphysema models (mean linear intercept, 78.1±2.5 μm vs 83.2±2.6 μm in elastase models and 75.6±1.4 μm vs 80.5±3.2 μm in smoke models, P<0.05). Furthermore, we showed that pioglitazone-pretreated ASCs increased vascular endothelial growth factor (VEGF) production both in vitro and in mouse lungs in the smoke-induced emphysema model. Pioglitazone-pretreated ASCs may have more potent therapeutic effects than non-pretreated ASCs in emphysema mouse models.

Aminoglycoside antibiotics including gentamicin (GM) induce delayed ototoxic effects such as hearing loss after long-term use, unlike the early-onset ototoxicity caused by cisplatin. The purpose of the study was to identify the mechanism of the delayed GM-induced ototoxicity by exploring the role of autophagy in vitro and in vivo. Treating HEI-OC1 auditory cells with GM led to a time-dependent increase of the autophagosome marker LC3-II, which was accompanied by cell death. In contrast, cisplatin and penicillin caused a rapid increase and had no effect on LC3-II levels, respectively. LC3-II-expressing autophagosomes co-localized with the labeled GM. GM-treated autophagosomes expressed reduced levels of Rab7, which is necessary for the fusion of autophagosomes with lysosomes. When the autophagic flux enhancer rapamycin was applied to GM-treated cells, Rab7 and the lysosomal enzyme cathepsin D were upregulated, and increased cell survival was observed. In animal studies, the intraperitoneal injection of GM worsened hearing thresholds and induced the accumulation of LC3 in the organ of Corti. This hearing impairment was attenuated by rapamycin. These findings suggest that the delayed onset-ototoxicity of GM may be closely related to the accumulation of autophagosomes via impaired autophagy. This GM-induced auditory cell death could be inhibited by enhancing autophagic flux.

Circulating tumor cells (CTCs) are rare cells and the presence of these cells may indicate a poor prognosis and a high potential for metastasis. Despite highly promising clinical applications, CTCs have not been investigated thoroughly, due to many technical limitations faced in their isolation and identification. Current CTC detection techniques mostly take the epithelial marker epithelial cell adhesion molecule (EpCAM), however, accumulating evidence suggests that CTCs show heterogeneous EpCAM expression due to the epithelial-to-mesenchymal transition (EMT). In this study, we report that a microchip filter device incorporating slit arrays and 3-dimensional flow that can separate heterogeneous population of cells with marker for CTCs. To select target we cultured breast cancer cells under prolonged mammosphere culture conditions which induced EMT phenotype. Under these conditions, cells show upregulation of caveolin1 (CAV1) but down-regulation of EpCAM expression. The proposed device which contains CAV1-EpCAM conjugated bead has several tens of times increased throughput. More importantly, this platform enables the enhanced capture yield from metastatic breast cancer patients and obtained cells that expressed various EMT markers. Further understanding of these EMT-related phenotypes will lead to improved detection techniques and may provide an opportunity to develop therapeutic strategies for effective treatment and prevention of cancer metastasis.

Nicotinamide phosphoribosyltransferase (NAMPT) is the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway and plays a crucial role in the maintenance of the NAD+ pool during inflammation. Considering that macrophages are essential for tissue homeostasis and inflammation, we sought to examine the functional impact of NAMPT in inflammatory macrophages, particularly in the context of inflammatory bowel disease (IBD). In this study, we show that mice with NAMPT deletion within the myeloid compartment (Namptf/fLysMCre+/-, Nampt mKO) have more pronounced colitis with lower survival rates, as well as numerous uncleared apoptotic corpses within the mucosal layer. Nampt-deficient macrophages exhibit reduced phagocytic activity due to insufficient NAD+ abundance, which is required to produce NADPH for the oxidative burst. Nicotinamide mononucleotide (NMN) treatment rescues NADPH levels in Nampt mKO macrophages and sustains superoxide generation via NADPH oxidase. Consequently, Nampt mKO mice fail to clear dead cells during tissue repair, leading to substantially prolonged chronic colitis. Moreover, systemic administration of NMN, to supply NAD+, effectively suppresses the disease severity of DSS-induced colitis. Collectively, our findings suggest that activation of the NAMPT-dependent NAD+ biosynthetic pathway, via NMN administration, is a potential therapeutic strategy for managing inflammatory diseases.

Cancer cells utilize epigenetic alterations to acquire autonomous capabilities for tumor maintenance. Here, we show that pancreatic ductal adenocarcinoma (PDA) cells utilize super-enhancers (SEs) to activate the transcription factor EVI1 (ecotropic viral integration site 1) gene, resulting in activation of an EVI1-dependent transcription program conferring PDA tumorigenesis. Our data indicate that SE is the vital cis-acting element to maintain aberrant EVI1 transcription in PDA cells. Consistent with disease progression and inferior survival outcomes of PDA patients, we further show that EVI1 upregulation is a major cause of aggressive tumor phenotypes. Specifically, EVI1 promotes anchorage-independent growth and motility in vitro and enhances tumor propagation in vivo. Mechanistically, EVI1-dependent activation of tumor-promoting gene expression programs through the stepwise configuration of the active enhancer chromatin attributes to these phenotypes. In sum, our findings support the premise that EVI1 is a crucial driver of oncogenic transcription programs in PDA cells. Further, we emphasize the instructive role of epigenetic aberrancy in establishing PDA tumorigenesis.

The programmed cell death pathway necroptosis may synergize with the DNA damage response (DDR) in opposing tumor progression. While our basic mechanistic understanding of the necroptotic cell death advances rapidly, its prognostic implications have not been thoroughly examined in cancers.